Developmental Dynamics of Naturalistic Social Behavior Revealed by Longitudinal Multi-Animal Tracking

Early-life social experiences with parents and littermates are critical for the development of social behavior and cognition in social species. Yet, how naturalistic social interactions evolve across development remains poorly understood, because reliable tracking of multiple freely interacting animals is challenged by frequent occlusion and body overlap. To address this challenge, we developed Move-Altogether (MovAl), a multi-animal pose estimation pipeline integrating video object segmentation and contour-guided preprocessing that improved identity stability and pose accuracy compared with widely used methods. Using MovAl-derived pose sequences, we established a denoising autoencoder (DAE)-based framework to map longitudinal home-cage behavior within a common three-dimensional space defined by inter-animal spacing, social orientation, and interaction dynamics. Within this space, control mice showed progressive reorganization of group behavior from 3 to 8 weeks of age, establishing a developmental reference for naturalistic social organization. Against this reference, prenatal valproic acid (VPA) exposure produced prominent alterations in social behavior organization at 3-5 weeks of age, demonstrating that the consequences of neurodevelopmental perturbation can be detected through changes in naturalistic group behavior among littermates. Extending the analysis to preweaning mother-pup interactions revealed increased maternal movement and reduced mother-pup proximity in the VPA condition, indicating alterations in the early social environment that preceded postweaning behavioral differences. These findings suggest that quantitative characterization of early social interactions may provide a means to identify early behavioral alterations associated with atypical neurodevelopment. Together, the MovAl-DAE framework enables longitudinal quantification of naturalistic social behavior, defining its developmental organization and revealing alterations associated with neurodevelopmental perturbation.